Hemostasis, i.e. the arrest of hemorrhage, is a mechanism which comprises essentially two consecutively functioning mechanisms: "primary hemostasis" is responsible for the immediate arrest of hemorrhage and is caused by the localization and aggregation of circulating platelets on damaged vascular surfaces or exposed tissues and subsequent formation of a thrombus; "secondary hemostasis" is responsible for the long-term arrest of hemorrhage and is caused by a chain of enzymatic reactions resulting eventually in the formation of fibrin.
Abnormalities of primary hemostasis are clinical conditions associated with bleeding tendencies which can become life-threatening in traumatic situations such as operation, delivery, invasive diagnostic and therapeutic procedures as well as traumatic injury. The evaluation of the degree of primary hemostasis is thus of a high clinical importance.
Several procedures have been hitherto employed in order to evaluate primary hemostasis. One procedure termed "the bleeding time test", which is the most common clinical test for determining primary hemostasis, is carried out by inducing a controlled cut in the arm of the tested subject and determining the duration before bleeding is arrested. This procedure has relatively low clinical relevance as it is able to identify only severe abnormalities in primary hemostasis and is thus unable to distinguish between normal subjects and those whose primary hemostasis is slightly defective. Furthermore, this method is virtually impossible to standardize since the duration of bleeding depends strongly on the precise size and location of the cut as well as on the venus and arterial blood pressures.
Platelet aggregation studies are usually performed in platelet-rich plasma (PRP) using a turbidometric device according to Born (Born G. V. R., Nature, 194, 927-929 (1962)). One drawback of turbidometric aggregometry is that the use of PRP necessitates centrifugation and separation of platelets from other blood cells which manipulation may alter platelets' properties and behavior. Another drawback of the turbidometric method is in that it is time consuming and laborious. Finally, the evaluation of such a test is limited by the optical quality of PRP which is affected by the levels of lipids in the plasma.
Platelets' aggregation was also tested in whole blood (WB) (Riess H., Am. J. Clin. Pathol, 85, 50-56, (1986)). According to this technique, platelet aggregation was measured by an increase in impedence across two electrodes placed in the blood sample. However, in this technique as well as the turbidometric technique described above, the platelets' aggregation was induced by artificial reagents and consequently the conditions in which the aggregation was measured were clearly non-physiological.
Several other procedures involved subjecting PRP to shear forces and determining platelet aggregation under these conditions. The shear forces in such tests were induced either by a cone-plate apparatus in which a rotating member was rotated within a cylindrical vessel, or by various sophisticated means adapted to produce a continuous flow of fluids (Tippe A. et al., Thrombosis Res., 67:407-418 (1992); Ikeda et al., J. Clin. Invest., 87, 1234-1240 (1991)); Fukiyama M. et al., Thrombosis Res. 64:253-260 (1989)). In such tests, which were generally conducted with PRP, the shear induced adhesions took place on the surfaces of the test vessel. The physiological significance of adhesion of platelets to artificial matrix under such conditions is questionable and this procedure indeed very often fails to provide clinically significant results. In addition, the means to produce the shear force in some of the procedures disclosed in the above publications were often complicated and expensive rendering the procedure unsuitable as a routine clinical procedure.
Another hitherto disclosed procedure involved determining adhesion of platelets, in a platelet rich plasma (PRP) (Vlodavksy, I. et al., Thrombosis Research, 18, 179-191, (1983)) and in whole blood (Lavee J. et a., The Journal of Thoracic and Cardiovascular Surgery, 97, (2), 204-212, (1989)) to an extracellular matrix (ECM).
It is the object of the invention lo provide a novel method and apparatus for evaluating platelets' function in primary hemostasis.